The present invention relates to novel preparations of osteogenic factors, methods for their isolation and uses thereof (to repair bone defects). The preparations so isolated exhibit the ability to promote or stimulate the formation of bone at the site of their application. Bone is a highly specialized connective tissue with unique mechanical properties derived from its extensive matrix structure. A network of fibrous bundles composed of the protein, collagen, is presumed to provide the tension-resistant behavior of bone. In addition, other materials including proteoglycans, noncollagenous proteins, lipids and acidic proteins associated with a mineral phase consisting primarily of poorly crystallized hydroxyapatite are deposited in the extensive matrix architecture of bone. Bone tissue is continuously renewed, by a process referred to as remodeling, throughout the life of mammals. This physiologic process might serve to maintain the properties of a young tissue.
The processes of bone formation and renewal are carried out by specialized cells. Osteogenesis vis-a-vis morphogenesis and growth of bone is presumably carried out by the "osteoblasts" (bone forming cells). Remodeling oi one is apparently broug by an interplay between the activities of the bone-resorbing cells called "osteoclasts" and the bone-forming osteoblasts. The boney skeleton is thus not only an architectural structure with a mechanical function but also is a living tissue capable of growth, modeling, remodeling, and repair. Since these processes are carried out by specialized living cells, chemical (pharmaceutical/hormonal), physical and physiochemical alterations can affect the quality, quantity and shaping of bone tissue.
A variety of pathological disorders as well as physical stress (for example, fracture) necessitate active formation of bone tissue at rate that are significantly higher than that which can be supported by the normal milieu of the body. It is thus of value to identify physiologically acceptable substances (hormones/pharmaceuticals/growth factors) that can induce the formation of bone at a predetermined site where such substrances are applied, for example, by inplantation. Such agents could either provide a permissive matrix structure for the deposition of bone-forming cells, or stimulate bone-forming cells, or induce the differentiation of appropriate progenitors of bone-forming cells.
The presence of proteinaceous and prostaglandin-like growth stimulators for osteoblasts has been examined, see reviewsi Raisz, L. G . et al., The New England Journal of Medicine, Vol. 309, No. 1, pp. 29-35 (1983) and Raisz, L. G., et al., The New England Journal of Medicine, Vol. 309, No. 2, pp. 83-89 (1983).
The observation that a bone graft from the same individual or a compatible individual leads to the formation of new healthy bone at the site of the graft, led to the hypothesis that bone contains active proteins which promote local osteogensis. Urist, et al. disclosed evidence that bone matrix-associated noncollagenous proteins can be isolated by dissociative treatment of demineralized bone powder and that this mixture of noncollagenous proteins contain the local osteoinductive capability which was designated by Urist (e.g., Science, Vol. 150, p. 893 (1965)) as bone morphogenetic activity.
A variety of osteogenic, cartilage-inducing and bone-inducing protein preparations have been described in the art. Urist, et al. and others have described various partially fractionated protein preparations with osteoinductive properties. These preparations are fractionated from the noncollagenous protein mixture extracted using different dissociative treatment of demineralized bone powder and subjecting the extract to various protein fractionation steps. Several such preparations have been characterized by different assays to determine their biological activities and by protein components identified using different standard protein analytical methods.
Urist, et al., Proceedings of The Society for Experimental Biology And Medicine, 162, pp. 48-53 (1979), disclosed isolation of bone morphogenetic protein (BMP) from demineralized rabbit bone matrix. The reference discloses that BMP appears to contain a multitude of major protein components having molecular weights in the range of between 94,000 daltons (94K) to less than 14,000 daltons (14K) based on reducing SDS polyacrylamide gel electrophoretic (SDS-PAGE) analysis.
Urist, et al. in Proc. Nat'l. Acad. Sci. USA, Vol. 76, No. 4, pp. 1828-1832 (April, 1979), disclosed another preparation of BMP obtained from demineralized rabbit bone matrix. Five protein fractions each characterized by having a major component with an apparent molecular weight of 94K, 68K, 43K, 21K and 14.3K were identified by subjecting these preparations to SDS polyacrylamide gel electrophoresis. All five protein preparations were eluted from a gel column with o-methylmannoside. Four of the five preparations, namely those with major components of molecular weights ranging from 68K to 14.3K were eluted with ethyleneglycol and two preparations, namely those with major components of molecular weights from 21K and 14.3K were precipitated with calcium phosphate. All three groups of eluates were found to have comparable BMP activity. The reference suggests that the BMP activity in the third group (the preparations characterized by major components of 21K and 14.3K proteins) may result from dissociation of a low molecular weight hydrophobic molecule carried by a glycoprotein. The reference suggests the alternative possibilities that BMP could be a single glycoprotein molecule, that the biologic activity may be a function of a protein aggregate or that BMP activity may not be associated with bone glycoprotein at all (pg. 1831).
Urist, U.S. Pat. No. 4,294,753, disclosed that the molecular weight of BMP may range between about 20K and 63K (col. 4, lines 45-61). The reference disclosed that BMP preparation isolated from rabbit dentin matrix protein mixture appears to have a major component with a molecular weight of about 23K. Because a protein fraction obtained from osteosarcoma cells has a molecular weight of 63K, it was suggested that the matrix free 63K protein may be a BMP precursor.
Hanamura and Urist, et al., Clin. Ortho. and Rel. Res. No. 153 232-240 (November-December (1980), disclosed the purification of osteosarcoma produced material with bone morphogenetic activity into three main fractions characterized by having a major component of molecular weight of 16K, 12.5K and 7K, respectively. Fractions characterized by a major component with a higher molecular weight including a 22K protein were observed during initial purification steps; active fractions purified from such preparations did not contain the 22K protein. Based on these results, the 12.5K and 16K proteins were tentatively identified as BMP.
Conover and Urist, et al., The Chemistry and Biology of Mineralized Connective Tissues, Elsevier North Holland, Inc., Arthur Veis, editor, pp. 597-606 (1981), discloses the isolation of a BMP fraction from demineralized rabbit dentin. Preparations containing proteins having average molecular weights of 30K, 23K, 18K, 15K and 12K were identified. While it was suggested that a 23K protein might represent the active BMP fraction, it was acknowledged that the active fraction might be the 18K, 15K or 12K proteins which they were unable to separate from the 30K and 23K fractions.
Farley, et al., Biochemistry, Vol. 21, No. 14, pp. 3502-3507 (1982), discloses purification of a skeletal growth factor from demineralized human bone matrix with an apparent molecular weight of 83K. The disclosure makes reference to a 1981 reference (Trans. Annu. Meet.- Orthop. Res. Soc., 6, 136 (1981)) by Urist, Conover and others, describing bone morphogenetic protein as having a molecular weight of 23K.
Urist, et al., Clin. Ortho. and Rel. Res., No. 162, pp. 219-232, discloses a low molecular weight bone morphogenetic protein fraction extracted from bovine bone matrix and fractionated by ion exchange and gel chromatography. The reference discloses that bovine BMP may consist of components ranging in molecular weight from 12K to 30K with the main components corresponding to molecular weights of 23K, 18K and 12K. The reference suggests that the 18K component is the active protein of the group because of its invariable presence in active fractions.
Urist, et al., Proc. Soc. Exp. Biol. and Med., 173, pp. 194-199 (1983), identifies human bone morphogenetic protein (hBMP) extracted from demineralized human bone matrix as an 18K molecular weight protein. The 18K protein was identified as putative hBMP as a result of its invariable presence in chromatographic fractions having high hBMP activity and general absence in those fractions lacking such activity. 34K, 24K and 14K protein components isolated from the demineralized bone were found not to induce bone formation.
Seyedin, et al., U.S. Pat. Nos. 4,434,094, and 4,627,982 describe the work in Urist, U.S. Pat. No. 4,294,753 and state that in the Urist patent, BMP was not fully characterized. The Seyedin patents describe a process for partially purifying an osteogenic factor and describe the factors as having a molecular weight of less than or equal to 30K.
Urist, et al., Science, 220, pp. 680-686 (1983), again identifies BMP purified from demineralized bone matrix as an 18K molecular weight protein. Variable quantities of 14K, 24K and 34K proteins were isolated with the 18K protein but the reference discloses that each of the last three protein fractions can be removed without loss of BMP activity. The reference states that the 18K fraction is responsible for BMP activity and suggests that the 34K, 24K and 14K proteins are individually inactive but are subunits of a larger BMP complex with the 18K protein.
Urist, et al., Proc. Nat'l. Acad. Sci. USA, 81, pp. 371-375 (1984), confirms that bovine BMP has an apparent molecular weight of 18.5K daltons. The publication further discloses other bone derived proteins with apparent molecular weights of 17.5K and 17K, proteins with higher molecular weights of 34K, 24K and 22K and a protein with a lower molecular weight of 14K. The publication provided the N-terminal sequence for the 17.5K protein which had an unblocked amino terminus.
Urist, European Patent Application No. 212,474, discloses peptide fragments having molecular weights between about 4K and 7K comprising at least an active portion of the osteoinductive and immunoreactive domain of the 17.5K BMP molecule.
Wang, et al., Pat. Cooperation Treaty Application No. WO 88/00205, claiming priority based on applications including U.S. Ser. No. 880,776 filed Jul. 1, 1986, discloses a bovine bone inductive factor which is isolated from demineralized bone powder by a procedure comprising a number of chromatographic and dialysis steps. The bone inductive factor so isolated was found to contain, as judged by a non-reducing SDS-PAGE analysis, one or more proteins having a molecular weight of approximately 28,000 to 30,000 daltons. Reducing SDS-PAGE analysis of the active protein(s) yielded two major bands having the mobility of proteins having molecular weights of 18,000 daltons and 20,000 daltons respectively. Wang, et al. discloses three bovine proteins designated BMP-1, BMP-2 and BMP-3 where BMP is bone morphogenetic protein and provides peptide sequences for the proteins. Wang, et al. also discloses the nucleotide sequences and amino acid sequences predicted thereby of four human proteins designated BMP-1, BMP-2 Class I, BMP-2 Class II and BMP-3.
Wozney, et al., Science 242, pp. 1528-1533 (1988), describes the nucleotide sequences and amino acid sequences predicted thereby of three human complementary DNA clones (designated BMP-1, BMP-2A and BMP-3) corresponding to three polypeptides present in an extract of bovine bone which is capable of inducing de novo bone formation. Recombinant human BMP-1, BMP-2A and BMP-3 proteins were said to be independently capable of inducing the formation of cartilage in vivo. The nucleotide sequence and derived amino acid sequence of a fourth complementary DNA clone (designated BMP-2B) is also described. The BMP-1, BMP-2A, BMP-2B and BMP-3 proteins of this publication appear to correspond, respectively, to the BMP-1, BMP-2 Class I, BMP-2 Class II and BMP-3 proteins, respectively, of Wang, et al.
Sen, U.S. Pat. No. 4,804,744 issued Feb. 14, 1989, discloses a preparation of an osteogenic protein which is a member of the P3 family of proteins and which has an apaprent molecular weight of 22,000 to 24,000 daltons as revealed by coomassie blue staining of reducing SDS-PAGE analysis.
Lyons, et al., Proc. Nat'l. Acad. Sci. USA 86, pp. 4554-4558 (1989), describes the nucleotide sequence and derived amino acid sequence of a complementary DNa clone (designated Vgr-1) encoding a mouse protein which contains homologous regions for the deduced amino acid sequences of BMP-2A, BMP-2B and BMP-3.
Luyten, et al., J. Biol. Chem. 264, pp. 13377-13380 (1989), describes the purification and partial amino acid sequence analysis of a polypeptide present in an extract of bovine bone said to be capable of inducing de novo bone formation. This protein, designated osteogenin, has an apparent molecular mass of 22,000 daltons as judged by reducing SDS-PAGE analysis, and an apparent molecular mass of 30,000 to 40,000 daltons as judged by a non-reducing SDS-PAGE analysis. The amino acid sequences reported for osteogenin are said to show considerable homology to BMP-3 as described by Wozney, et al.
Bentz, et al., J. Bone and Mineral Res., 4 Supplement 1, p. S280 No. 650 (1989) and Bentz, et al., J. Cell Biol., 107, 162a No. 918 (1989) describe a protein material isolated from demineralized bone matrix said to promote osteoinduction in the rat. The osteoinductive factor (OIF) was identified as a glycoprotein and was said to exhibit osteoinductive activity only in the presence of TGF-.beta.1 or TGF-.beta.2. OIF had an apparent molecular mass of 22,000 to 28,000 daltons based on SDS gel electrophoresis and was identified as a monomeric molecule in light of the fact that reduction does not alter its mobility on SDS-PAGE.